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What's template got to do with it?

As an investigator, you set a high bar for data quality and assay performance. Running clean, efficient and reproducible assays requires protocol optimization, high-quality reagents and well-trained researchers. But even the most technically sound experiments rely on the use of high-quality starting materials to produce the best results.
This is especially true for RT-qPCR assays where poor quality template DNA can impair reproducibility, dampen the signal-to-noise ratio and even create misleading results. In this article, we will discuss the importance of starting with a high-quality cDNA template for your RT-qPCR experiments and our top tips for how to do so.

High-quality RNA extraction
We can't emphasize the importance of this enough. Isolating high-quality RNA is the most critical step for the successful preparation of your cDNA library, as it requires performing efficient and reproducible RT-qPCR assays as well as microarray studies and high-throughput transcriptome analyses using next-generation sequencing techniques. Therefore, it is essential that you get the most from your RNA isolation procedure. Top-quality experiments require top-quality starting material; thus, maximizing the yield of non-degraded RNA during extraction is key.

For instance, while organic extraction techniques with homogenization in a phenol-containing solution can be simple to perform and readily scaled for larger samples (such as human or animal tissues), they are not very amenable to high-throughput processing and can be difficult to automate. Therefore, given their more laborious nature, kit-based strategies such as spin column extraction, may be more efficient and reproducible. This strategy employs a solid phase extraction technique to bind and isolate RNA within filter-based spin columns.

The Roche High Pure RNA Isolation Kit is ideal for low- to medium-throughput assays for mini scale RNA isolation. In this method, nucleic acids bind to the surface of the glass fiber fleece in the presence of a chaotropic salt (guanidine HCl), allowing the High Pure filter to specifically immobilize nucleic acids (both DNA and RNA), while contaminants are removed by centrifugation. DNA is digested with DNase I directly on the filter, and the concentrated RNA is eluted in a small volume of low-salt buffer. The main advantage of this technique is its simple and straightforward protocol and availability in kit format (great for novice researchers). Multiple samples can be processed quickly and with high sensitivity, specificity and reproducibility for downstream experiments. The advantages of this strategy are not limited to newly obtained samples either, as the High Pure FFPET RNA Isolation Kit is designed for RNA isolation and purification from formalin-fixed, macrodissected or biopsy samples, as well as paraffin-embedded tissue via a deparaffinization procedure.

However, if you plan to perform automated RNA extraction, magnetic particle extraction may be the most efficient and amenable to automation. This method of bioseparation utilizes beads with a paramagnetic core coated with a matrix of silica (most commonly) for binding nucleic acids. In this strategy, the magnetic beads can be quickly collected by being placed in proximity to an external magnetic field, and is therefore the RNA isolation technique of choice for automated protocols, as the magnetic collection and resuspension steps are rapid and simple to perform. This method can be employed over a range of throughput levels.

The Roche MagNA Pure Compact System is an automated benchtop instrument for processing one to eight samples per run, ideal for low- to medium-sample throughput as well as versatile nucleic acid purification needs. For higher volume needs, the MagNA Pure LC 2.0 tabletop instrument can process up to 32 different samples in a single run using an automated robotic system for isolating any type of nucleic acid in as little as 60 minutes. For maximum automated efficiency, the Roche MagNA Pure 96 System can process up to 96 samples for purification of DNA, RNA, or viral nucleic acids from a wide array of starting materials in less than one hour using barcoded, prefilled trays with ready-to-use reagents. Minimal lot-to-lot variation ensures excellent reproducibility and precision over a broad linear range. The resulting nucleic acids are suitable for a vast range of downstream applications.

Robust cDNA library preparation
After extracting high-quality RNA, the next critical step is the high-yield synthesis of your cDNA library. When performing two-step RT-qPCR, the generation of cDNA can be done using oligo dT primers, random hexamers, gene-specific primers or even of mixture of these. The subsequent cDNA can be stably stored for future analysis, enabling tremendous flexibility for optimization of PCR conditions and the ability to perform future amplification reactions with the remaining cDNA. Therefore, it is essential to create a robust, efficient and accurate cDNA library for your experimental needs. For optimal priming, oligo dT primers can be utilized to generate full-length cDNAs by binding at the beginning of poly(A) tails. Random hexamers can also be used for more uniform representation of all RNA sequences by priming throughout the length of RNA molecules, thereby allowing reverse transcription of RNAs that do not harbor a poly(A) tail. Gene-specific primers provide high specificity and reliability and are the employed method for one-step RT-qPCR reactions. However, they remain limited in flexibility compared to oligo dT or random hexamers, which can be suboptimal when analyzing precious tissue or cell samples. Each of these library preparation options are available within commercial cDNA synthesis kits.

Importantly, the use of high-quality cDNA synthesis kits can mean optimized compatibility with your specific real-time PCR instrument. This results in more accurate linear quantification over a wide range of template dilutions, thereby enabling reliable analysis of both low- and high-abundance transcripts. Additionally, optimized cDNA synthesis kits can enhance robustness, permitting transcription of more difficult targets, such as GC-rich RNA with high secondary structures, with high reproducibility.
Performing high-yield, efficient and reliable nucleic acid extraction and cDNA library preparation methods are key to creating successful, high-quality RT-qPCR data. So we ask, what does template have to do with it? Well, pretty much everything.

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